Both gain-of-function and loss-of-function de novo CACNA1A mutations cause severe developmental epileptic encephalopathies in the spectrum of Lennox-Gastaut syndrome
Both gain-of-function and loss-of-function de novo CACNA1A mutations cause severe developmental epileptic encephalopathies in the spectrum of Lennox-Gastaut syndrome
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DOI：
10.1111/epi.16316
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发表时间：
2019-08-29
期刊：
影响因子：
5.6
通讯作者：
Rossignol, Elsa
中科院分区：
文献类型：
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作者：
Jiang, Xiao;Raju, Praveen K.;Rossignol, Elsa
目的发育性癫痫性脑病（dei）是一种遗传异质性的儿童期严重癫痫，伴有发育迟缓或认知缺陷。在这项研究中，我们探讨了dee相关的CACNA1A基因新生突变的致病机制。方法我们研究了四种新的dee相关的CACNA1A突变对功能的影响，包括先前描述的p.A713T变异和三种新变异（p.a 1396m, p.G230V和p.a 1357s）。在HEK293细胞中表达突变cdna，并进行全细胞电压钳记录，以检测其对Ca(V)2.1通道功能的影响。利用免疫荧光显微镜和三维（3D）建模评估通道定位和结构。结果我们发现G230V和I1357S突变导致全细胞电流密度降低和细胞膜通道表达降低的功能丧失效应。相比之下，A713T和V1396M变异导致功能获得效应，增加全细胞电流和促进电流激活（超极化移位）。A713T型也导致较慢的电流衰减。三维建模预测了有利于A713T和V1396M通道打开的构象变化。我们的研究结果表明，功能获得和功能丧失的CACNA1A突变都与类似的严重的dei相关，需要功能验证来阐明潜在的分子机制并指导治疗。
Objective Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene. Methods We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on Ca(V)2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling. Results We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M. Significance Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.